Dmc1在南方鲇卵母细胞减数分裂前期I中的作用机制研究

Meiotic prophase I is one of the key stages of oogenesis, and in this stage many genes are activated and /or silenced in a timely and spatially - coordinated manner so as to ensure correct development of prophase I. Dmc1 is a meiosis-specific gene that is a vital factor in the process of homologous recombination. In the previous studies, we successfully cloned the Dmc1 gene from the Southern catfish. Spatiotemporal expression analysis and transcriptome data showed that the Dmc1 gene has the highest expression at the meiosis stage. Thus we speculate that Dmc1 may be critical for the development of meiotic prophase I of oogenesis. In order to test our hypothesis, In vivo gene knockout (loss of function) and overexpression (gain of function) experiments will be employed first to determine the effects of Dmc1 on the development of prophase I, and then the Dmc1 target will be screened using Chip-seq and several of these target genes will be selected to testify by EMSA. Our study aims to clarify the critical roles of Dmc1 for the development of meiotic prophase I of oogenesis and molecular mechanisms underlying this process, thus providing theoretical basis for the breeding of fish in aquaculture.

卵母细胞减数分裂前期I是卵子发生过程中的关键阶段，多种不同类型的基因按时空顺序发生转录激活或沉默是确保减数分裂前期I正常进行的必要条件。Dmc1是减数分裂期特异性表达基因，是同源重组过程中的核心因子。通过前期研究，我们成功分离并克隆了南方鲇Dmc1基因；时空表达分析和转录组数据均表明，Dmc1基因在减数分裂期表达量最高。由此推测Dmc1可能在减数分裂前期I发挥重要作用。基于上述假设，本课题首先利用基因敲除和过表达技术观察南方鲇Dmc1对减数分裂前期I进程的影响；然后利用染色质免疫共沉淀–DNA测序技术筛选卵母细胞内与Dmc1结合的靶基因，并通过EMSA验证重要靶基因。通过相关研究旨在阐明Dmc1在减数分裂前期I的功能和分子机制，为水产养殖鱼类遗传育种提供理论基础。

卵子形成需要经过减数分裂，减数分裂过程中同源染色体要进行配对、联会、重组等一系列关键事件，以保障遗传物质的均等分离。减数分裂同源重组发生在前期 I，是减数分裂关键中的关键，因为同源染色体能否精确分离取决于重组的完成与否。Dmc1是同源重组过程中的核心因子。目前在硬骨鱼类中，关于Dmc1的研究大多都集中在表达层面，而其在同源重组过程中的作用机制研究甚少。有鉴于此，课题组运用CRISPR-Cas9技术、基因过表达技术、Chip-seq技术、生物信息学方法和EMSA实验研究其功能和作用机制。结果发现，55dah时，只有对照组中的南方鲇已进入减数分裂，并且突变组中Spo11和Scp3的表达量显著低于对照组（p<0.05），表明突变Dmc1可以推迟南方鲇卵巢中的生殖细胞进入减数分裂；筛选出拟定靶片段754条，进一步验证发现，Dmc1确与选取的三条靶片段相互作用。本研究可为后续构建Dmc1在同源重组中的调控网络提供依据，同时为南方鲇人工繁殖生产高质量卵子提供理论基础。